The stress-responsive mitochondrial sirtuin SIRT4 controls cellular energy metabolism in a NAD+-dependent manner and is implicated in cellular senescence and aging. Here we reveal a novel function of SIRT4 in mitochondrial morphology/quality control and regulation of mitophagy. We report that moderate overexpression of SIRT4, but not its enzymatically inactive mutant H161Y, sensitized cells to mitochondrial stress. CCCP-triggered dissipation of the mitochondrial membrane potential resulted in increased mitochondrial ROS levels and autophagic flux, but surprisingly led to increased mitochondrial mass and decreased Parkin-regulated mitophagy. The anti-respiratory effect of elevated SIRT4 was accompanied by increased levels of the inner-membrane bound long form of the GTPase OPA1 (L-OPA1) that promotes mitochondrial fusion and thereby counteracts fission and mitophagy. Consistent with this, upregulation of endogenous SIRT4 expression in fibroblast models of senescence either by transfection with miR-15b inhibitors or by ionizing radiation increased L-OPA1 levels and mitochondrial fusion in a SIRT4-dependent manner. We further demonstrate that SIRT4 interacts physically with OPA1 in co-immunoprecipitation experiments. Overall, we propose that the SIRT4-OPA1 axis is causally linked to mitochondrial dysfunction and altered mitochondrial dynamics that translates into aging-associated decreased mitophagy based on an unbalanced mitochondrial fusion/fission cycle.

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Cytokine-induced signal transduction is executed by natural biological switches, which among many others control immune-related processes. Here, we show that synthetic cytokine receptors (SyCyRs) can induce cytokine signaling using non-physiological ligands. High-affinity GFP- and mCherry-nanobodies were fused to transmembrane and intracellular domains of the IL-6/IL-11 and IL-23 cytokine receptors gp130 and IL-12Rβ1/IL-23R, respectively. Homo- and heterodimeric GFP:mCherry fusion proteins as synthetic cytokine-like ligands were able to induce canonical signaling in vitro and in vivo. Using SyCyR ligands, we show that IL-23 receptor homodimerization results in its activation and IL-23-like signal transduction. Moreover, trimeric receptor assembly induces trans-phosphorylation among cytokine receptors with associated Janus kinases. The SyCyR technology allows biochemical analyses of transmembrane receptor signaling in vitro and in vivo, cell-specific activation through SyCyR ligands using transgenic animals and possible therapeutic regimes involving non-physiological targets during immunotherapy.

Hepatic stellate cells (HSCs) were recently identified as liverresident mesenchymal stem cells. HSCs are activated after liver injury and involved in pivotal processes, such as liver development, immunoregulation, regeneration, and also fibrogenesis. To date, several studies have reported candidate pathways that regulate the plasticity of HSCs during physiological and pathophysiological processes. Here we analyzed the expression changes and activity of the RAS family GTPases and thereby investigated the signaling networks of quiescent HSCs versus activated HSCs. For the first time, we report that embryonic stem cell-expressed RAS (ERAS) is specifically expressed in quiescent HSCs and down-regulated during HSC activation via promoter DNA methylation. Notably, in quiescent HSCs, the high level of ERAS protein correlates with the activation of AKT, STAT3, mTORC2, and HIPPO signaling pathways and inactivation of FOXO1 and YAP. Our data strongly indicate that in quiescent HSCs, ERAS targets AKT via two distinct pathways driven by PI3K␣/␦ and mTORC2, whereas in activated HSCs, RAS signaling shifts to RAF-MEK-ERK. Thus, in contrast to the reported role of ERAS in tumor cells associated with cell proliferation, our findings indicate that ERAS is important to maintain quiescence in HSCs.

The pro-inflammatory cytokine IL-23 is composed of the IL-12p40 subunit and p19 (1). IL-23 is a key factor for the development of T H 17 cells (2), which control antimicrobial and antifungal responses, but is also critically involved in the pathogenesis of chronic inflammatory disorders (3). The receptor complex is composed of the common IL-12 receptor ␤1 (IL-12 ␤1), 2 shared with IL-12, and the unique IL-23 receptor (4, 5).Single nucleotide polymorphisms within the IL23R gene were associated with various autoimmune diseases and the risk to develop cancer (1). Upon recruitment of the receptors by IL-23, which results in a noncanonical receptor complex formation (6), signaling is initiated by activation of associated Tyk2 (tyrosine kinase 2) and Jak2 (Janus kinase 2), which phosphorylate predominantly STAT3, and to a lesser extent STAT1, STAT4, and STAT5 (5). Recently, a noncanonical tyrosine-independent STAT3 activation site within the IL-23R was identified (7). In addition to STAT proteins PI3K, MAPK and NF-B signaling pathways were activated (7, 28). Ectodomain shedding of membrane-bound proteins leads to receptor protein down-regulation on the cell surface and the generation of soluble protein ectodomains with agonistic or antagonistic properties. Members of the ADAM (A disintegrin and metalloprotease) gene family are major ectodomain shedding proteinases. ADAM17 and its close relative ADAM10 are the major sheddases of this family, (8), with extensive overlap and compensation for several substrates, including EGF receptor ligands, TNF, TNF receptor, and IL-6R (9, 10). Activation of ADAM proteases is achieved by different stimuli including phorbol ester (phorbol-12-myristate-13-acetate (PMA)), ionomycin, ligands of G-protein-coupled receptors, ATP, bacterial toxins, bacterial metalloproteinases, and apoptosis (8). For some ADAM target proteins such as Notch, induction of intracellular signaling by the remaining intracellular domain cleavage product has been described (11). Previously, it was shown that alternative splicing of IL-23R result in a series of truncated soluble .Here, we discovered murine and human IL-23R as novel substrates of ADAM10 and ADAM17, resulting in the release of soluble IL-23R proteins, which retained their ability to bind to IL-23. Distinct areas within the murine and human IL-23R, which are important for ectodomain shedding, were identified in murine and human IL-23R. Immunoprecipitation analysis revealed domains 1 and 3 of IL-23R as critical ADAM17 interaction sites. Thus, we propose that ectodomain shedding is a second mechanism that contributes to the generation of soluble IL-23R variants.